A Thiamine-Dependent Enzyme Utilizes an Active Tetrahedral Intermediate in Vitamin K Biosynthesis

Article abstract of DOI:10.1021/jacs.6b03437

Enamine is a well-known reactive intermediate mediating essential thiamine-dependent catalysis in central metabolic pathways. However, this intermediate is not found in the thiamine-dependent catalysis of the vitamin K biosynthetic enzyme MenD. Instead, an active tetrahedral post-decarboxylation intermediate is stably formed in the enzyme and was structurally determined at 1.34 ? resolution in crystal. This intermediate takes a unique conformation that allows only one proton between its tetrahedral reaction center and the exo-ring nitrogen atom of the aminopyrimidine moiety in the cofactor with a short distance of 3.0 ?. It is readily convertible to the final product of the enzymic reaction with a solvent-exchangeable proton at its reaction center. These results show that the thiamine-dependent enzyme utilizes a tetrahedral intermediate in a mechanism distinct from the enamine catalytic chemistry.

Full text of DOI:10.1021/jacs.6b03437

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Communication
A thiamine-dependent enzyme utilizes an active
tetrahedral intermediate in vitamin K biosynthesis
Haigang Song, Chen Dong, Mingming Qin, Yaozong Chen,
Yueru Sun, Jingjing Liu, Wan Chan, and Zhihong Guo
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b03437 • Publication Date (Web): 23 May 2016
Downloaded from http://pubs.acs.org on May 28, 2016
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A thiamine-dependent enzyme utilizes an active tetrahedral inter-
mediate in vitamin K biosynthesis
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‖
⊥
Haigang Song , †,‡, Chen Dong,†, Mingming Qin,†, ‡ Yaozong Chen,†, ‡ Yueru Sun,†, ‡,┬ Jingjing
Liu,§ Wan Chan,†,§ Zhihong Guo*†, ‡
†Department of Chemistry, ‡State Key Laboratory for Molecular Neuroscience, §Environmental Science Program,
The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China.
Supporting Information Placeholder
ABSTRACT: Enamine is a well-known reactive intermediate
mediating essential thiamine-dependent catalysis in central
(a)
OH
OH
O
O
CO2
O
O
CO2
MenD
metabolic pathways. However, this intermediate is not found
in the thiamine-dependent catalysis of the vitamin K biosyn-
thetic enzyme MenD. Instead, an active tetrahedral post-
decarboxylation intermediate is stably formed in the enzyme
and structurally determined at 1.34 Å resolution in crystal.
This intermediate takes a unique conformation that allows
only one proton between its tetrahedral reaction center and
the exo-ring nitrogen atom of the aminopyrimidine moiety in
the cofactor with a short distance of 3.0 Å. It is readily con-
vertible to the final product of the enzymic reaction with a
solvent-exchangeable proton at its reaction center. These
results show that the thiamine-dependent enzyme utilizes a
tetrahedral intermediate in a mechanism distinct from the
enamine catalytic chemistry.
+
O2C
CO2
CO2
CO2
ThDP
CO2
SEPHCHC
(b)
N
N
1'
N
NH2
1'
N
NH2
4'
4'
Acyl anion
Enamine
OH
2α
N
2
OH
2α
N
2
2-O3P-O3POCH2CH2
S
2-O3P-O3POCH2CH2
S
CO2
CO2
6
(c)
MenD
MenD+ThDP
4
2
0
295
305
315
325
335
345
355
365
-2
-4
-6
Thiamine diphosphate (ThDP)-dependent enzymes cata-
lyze a broad range of reactions in diverse biological process-
es. Their catalysis involves activation of the ThDP cofactor as
an ylide or carbene and formation of a planar enamine in-
termediate.1 The reactive enamine intermediate has been
successfully captured in crystal in several thiamine enzymes
Wavelength (nm)
Figure 1. The thiamine disphophate-mediated reaction in
vitamin K2 biosynthesis. (a) The MenD-catalyzed reaction.
(b) The previously proposed enamine-acyl anion intermedi-
ate. (c) Circular dichroism spectrum of the intermediate. The
solution contained 2 mM α-ketoglutarate (2KG), 33 μM
MenD, 200 μM ThDP and 2 mM MgSO4 in 100 mM potassi-
um phosphate buffer at pH 7.0 and the control solutions
contained the enzyme with or without ThDP at the same
concentration.
2-5
and is generally accepted to be essential in the catalytic
process. In the last two decades, although some atypical
enamine tautomers have been suggested to play a role in
catalysis of a few ThDP-dependent enzymes, the central role
of enamine is not changed in thiamine catalysis.6-9
(1R, 2S, 5S, 6S)-2-succinyl -5-enolpyruvyl-6-hydroxy-3-
cyclohexene-1-carboxylate (SEPHCHC) synthase, or MenD, is
a ThDP-dependent enzyme catalyzing a distinctive Stetter-
like 1,4-addition reaction in bacterial biosynthesis of vitamin
K2 (Fig. 1a and Fig. S1).10, 11 Previous structural studies showed
that it is not significantly different from other ThDP-
dependent enzymes in active site architecture, cofactor bind-
ing, or overall three-dimensional structure, suggesting the
use of the canonical enamine intermediate in its catalysis
(Fig. 1b).12, 13 However, the post-decarboxylation intermediate
formed in situ presents a circular dichroism spectrum that is
clearly different from that of the enamine intermediate of
yeast transketolase (Fig. 1c and the supporting information).2
To understand the reaction intermediate, we determined
the crystal structures of the ThDP-bound E. coli MenD
soaked in 10 mM α-ketoglutarate solution at room tempera-
ture for 21 s, 2 min, 15 min and 90 min to a resolution of
1.34−2.3 Å by molecular replacement (Table S1). The resulting
structures (Fig. S2) overlap very well with a root mean square
deviation (r.m.s.d.) less than 0.2 Å over all comparable back-
bone carbon atoms and each protein subunit contains an
essentially identical cofactor-bound intermediate with well-
defined electron density (Fig. 2a and Fig. S3). This stable
intermediate contains a planar thiazolium ring with a C2-
appendage derived from succinic semialdehyde (Fig. 2a). Its
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The unique C2α stereochemistry and short C2α to N4’ dis-
C2α atom is clearly tetrahedral and its terminal carboxyl
group is firmly fixed in the active
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tance significantly affect protonation states of the C2α and N4’
atoms in the covalent intermediate. Out of the four possible
models with a different protonation pattern at these two
atoms (Fig. 2c), the first two (I and II) are strongly favored
due to the absence of steric repulsion by having only one
proton between C2α and N4’, whereas the other models are
disfavored due to overlapping of the van der Waals surface of
the two hydrogen atoms between these two atoms. In models
I-III, N4’ is constrained to have all its associated protons on
the same plane of the pyrimidine ring because of heavy ener-
gy penalty for breaking this co-planarity.18 In Model IV, this
constraint is removed to reduce but unable to eliminate the
steric repulsion. Both intermediates III and IV are similar to
the off-pathway protonation products of the enamine/acyl
anion intermediate in catalysis of many other ThDP-
dependent enzymes and are expected to be dissociated at the
C2-C2α bond to release succinic semialdehyde like the latter
in aqueous solution.1 However, Palmer and coworkers
showed that no succinic semialdehyde was released for at
least 30 min from the intermediate formed between ThDP-
bound MenD and α-ketoglutarate,19 further negating the
likelihood of the protonated Model III or IV.
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Although the crystal structure is unable to determine the
protonation state at C2α and N4’ at the current resolution (1.34
Å), the four possible models could be differentiated by their
chemical reactivity and proton exchangeability. Model I is
the typical acyl anion tautomer with a potential amine-to-
carbanion hydrogen bond, while Model II could be consid-
ered to take a near attack position to allow deprotonation of
C2α by the neutral imine, particularly when primed by iso-
chorismate, the second MenD substrate. These two different
forms of the intermediate may interconvert and exist in equi-
librium with the proton shuttling between C2α and N4’. Thus,
both Models I and II are active, on-pathway intermediates
with an exchangeable C2α proton. In contrast, both III and IV
are off-pathway intermediates with neither catalytic activity
nor exchangeable C2α proton because they are variants of the
protonated acyl anion tautomer.
Figure 2. Crystal structure of the tetrahedral intermediate
complexed with MenD at 1.34 Å. (a) Stereo diagram of the
2mFo-DFc electron density map of the tetrahedral intermedi-
ate contoured at 2.0σ in blue mesh. (b) Stereo diagram of the
tetrahedral intermediate bound to the active site. (c) Four
possible models of the tetrahedral intermediate with differ-
ent protonation states at C2α and N4’. The intermediate is
represented in sticks with grey carbon atoms, while the con-
served active site residues are represented in sticks with
green carbon atoms and the manganese (II) ion is denoted
with a magenta sphere in (b). The broken lines denote hy-
drogen bonds or metal-chelating bonds with a distance ≤ 3.6
Å in (b) and the green spheres denote the van der Waals
surface of protons between C2α and N4’ in (c). Only the hy-
drogen atoms on either C2α or N4’ are indicated by green
sticks in (c) with the dotted lines indicating the shortest in-
teratomic distance while those attached to other atoms are
omitted for simplicity.
To directly determine the solvent exchangeability of the
proton associated with C2α, the putative enzyme-bound in-
termediate was freshly prepared and mixed with one equal
volume of deuterated water. The exchange reaction was
quenched after 30 minute incubation at room temperature
and the succinic semialdehyde-ThDP adduct was isolated by
HPLC. The resulting adduct was found to produce two mo-
lecular ions at m/z 527 and 528 with an intensity ratio of 100 :
96.6 compared to 100 : 22.5 for the non-deuterated control
(Fig. 3a and Fig. S5), indicating 39.8% deuterium incorpora-
tion. In its NMR spectrum (Fig. 3b), the chemical shift of its
C2α-H was determined at δ5.31 ppm by comparison to that of
the C2α-protons in similar ThDP adducts such as 2-(1-
hydroxyethyl)-ThDP.20 The intensity of this C2α-H signal is
decreased by 32.8% compared to the non-deuterated adduct
after normalization with the intensity of the C6’ proton on
the aminopyrimidine ring at 7.30 ppm,21 while those of other
protons are not significantly changed (Fig. S6). The results
from mass spectrometry and NMR exhibit difference due to
uncertainties in NMR intensity integration but are roughly
consistent, demonstrating that deuteration occurs at C2α
with an exchange rate in the range of 65.8% (NMR result) to
79.6% (MS result) under the given conditions (maximum
site by salt bridges and hydrogen bonds with the conserved
residues Arg395 and Arg413 (Fig. 2b).
In the covalent intermediate, all the bond lengths and an-
gles of its planar thiazolium ring are comparable to those of a
similar enzyme-free ThDP adduct (Fig. S4).14 Its C2-C2α bond
is a typical σ bond with a bond length of 1.49 Å (Table S2)
that is comparable to that of the enzyme-bound ThDP or 3-
deaza-ThDP adducts with a tetrahedral and protonated C2α
atom.5, 15-17 Importantly, the C2α to N4’ distance is short (3.03
Å) and the C2α atom takes an S-configuration to position one
of its bonds in the direction of the N4’ atom. In comparison,
the C2α atom of other enzyme-bound adducts always takes a
configuration to allow the C2α-OH to form a hydrogen bond
with N4’ and thus prevent any of the C2α bonds from spatially
colliding with it (Table S2), despite the fact that the C2α to N4’
distance is also short (3.3-3.8 Å).5, 15-17
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deuterium incorporation = 50%). These experiments clearly
show that C2α is associated with an exchangeable proton in
the post-decarboxylation intermediate, rendering support to
its identity as either Model I or II.
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Figure 3. C2α-H exchangeability and activity of the putative intermediate. (a) ESI-MS molecular ion of the isolated succinic semi-
aldehyde-ThDP adduct after 30 min deuterium exchange at room temperature. The insert shows the molecular ion of the non-
deuterated adduct from the control reaction. (b) Partial 1H NMR spectra of succinic semialdehyde-ThDP purified from the deu-
terium-exchange reaction (red line) and the control reaction (black line). The intensity of C2α-H at δ5.31 is normalized with the
intensity of C6’-H at δ7.30 and provided below the corresponding peaks. The peak with an asterisk at δ5.51 belongs to C7’-H of
free ThDP. (c) Conversion of the putative intermediate to SEPHCHC in solution. SEPHCHC is converted to SHCHC by MenH for
HPLC detection using NAD+ as an internal control. The yield is indicated in percentage after normalization to the positive con-
trol. (d) Gradual disappearance of the succinic semialdehyde group of the intermediate in crystal. Shown is the 2mFo-DFc elec-
tron density map contoured at 1.0σ in blue mesh after soaking in 250 μM isochorismate at room temperature for the indicated
time
period.
To test its activity, the putative intermediate was first pre-
observations unambiguously demonstrate that the putative
intermediate is also active in crystal.
pared in solution and allowed to react with excess isochoris-
mate in the presence of the menaquinone biosynthetic en-
zyme MenH,22 which converted the SEPHCHC product to
Collectively, all the biochemical and crystallographic re-
sults support that the trapped ThDP-bound intermediate is
an active, on-pathway intermediate in the MenD catalysis
and takes a structure either as the acyl anion (Model I) or as
its near attack variant (Model II). More likely, the intermedi-
ate exists in both structural forms in an equilibrium that is
dominated by Model II due to its much higher stability (Fig.
4). In this structural model of the intermediate (Fig. 2c), the
protonated C2α is activated by the neutral iminopyrimidine of
the cofactor, similar to the activation of C2-H of the ThDP
cofactor.24 In both cases, N4’ of the cofactor and the reaction
center at either C2 or C2α are coerced into a short distance to
allow only one proton between them and to force the cofac-
tor to take the iminopyrimidine form. In connection to this
activation mechanism, it is interesting to note that C2α-H and
C2-H are comparable in pKa.25
stable
2-succinyl-6-hydroxy-2,
4-cyclohexadiene-1-
carboxylate (SHCHC) for quantitative measurement by
HPLC. Compared to the control reaction, the single turnover
experiment produced 94%, 78%, and 62% of SHCHC when
the intermediate was freshly prepared (t = 0) or stored at
room temperature for 5 min or 30 min before reaction with
isochorismate, respectively (Fig. 3c). The lower product levels
relative to the control may be due to inactivation of the puta-
tive intermediate by side reactions such as oxidation,23 but
nonetheless unequivocally demonstrates that it is active in
solution. Subsequently, the putative intermediate was pre-
pared in crystal by soaking and subjected to a second soaking
in isochorismate solution at room temperature. In the result-
ing crystal structures with a resolution from 1.60 to 2.24 Å,
the electron density of the C2 appendage gradually decreases
in quality with increasing soaking time, ranging from essen-
tially unchanged at 2 min to complete disappearance at 13
min (Fig. 3d), while other parts of the intermediate or the
polypeptide chains remain unchanged. In all cases,
SEPHCHC was detected in the soaking solution with a cou-
pled assay22 but not found in the solved structures, indicating
that it quickly exited the active site after formation. These
As noticed earlier, the MenD intermediate is formed due to
the special stereochemical environment at C2α that leads to
collision between C2α and N4’, whereas a C2α-OH to N4’ hy-
drogen bond is formed to avoid unfavorable interactions in
known enzyme-bound tetrahedral adducts of ThDP or its 3-
deaza analog.5, 15-17 From this difference, prevention of the
C2α-OH to N4’ hydrogen bond is suggested to be the key to
forming the tetrahedral intermediate in MenD. This is appar-
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Page 4 of 6
ently achieved by conformational control of the C2-
Corresponding Author
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appendage via strong interaction of its terminal carboxylate
with the conserved Arg395 and Arg413 (Fig. 2b). This confor-
mational control is thus believed to be the point where
MenD diverges from other ThDP-dependent enzymes (Fig.
4). In its absence, the ThDP-bound intermediate naturally
falls to an energy minima after decarboxylation by taking the
energetically favorable enamine structure and simultaneously
forming the C2α-OH to N4’ hydrogen bond in most known
ThDP-dependent
Zhihong Guo, E-mail: chguo@ust.hk.
Present Addresses
‖ School of Chemistry, University of St Andrews, North
Haugh, St Andrews, KY16 9ST, Scotland, UK.
⊥Department of Chemistry, University of Washington, Bagley
Hall 425, Box 351700, Seattle, Washington 98195-1700, USA.
┬Section of Structural Biology, Department of Medicine, Im-
perial College London, South Kensington SW7 2AZ, UK.
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2-
R1 =-CH2CH2OPO3-PO3
hydrogen-bonding
Arg395
O
Arg395
O
Arg413
O
Arg395
O
Arg413
O
Arg413
O
Notes
The authors declare no competing financial interests.
hydrogen bond/salt bridge
H
OH
S
OH
OH
MenD
OOC
H
N
R1
N
H
H
N
H
S
S
ACKNOWLEDGMENT
H
S
R1
R1
N
N
N
R1
N
H
α-KG
N
N
N
N
N
N
N
N
N
We thank Shanghai Synchrotron Radiation Facility (SSRF)
and National Center for Protein Science Shanghai (NCPSS)
for access to the beamlines BL17U and BL19U and the on-site
technical support. This work was supported by GRF601413
and N_HKUST621/13 from the RGC and SBI14SC05 from the
UGC of the HKSAR government. The coordinates and struc-
ture factors of the MenD complexes have been deposited in
the Protein Data Bank with accession codes 5EJ4, 5EJ5, 5EJ6,
5EJ7, 5EJ8, 5EJ9, and 5EJA.
activated ThDP
H
H
Intermediate II
Intermediate I
C2-C2α
xrotation
Arg395
Arg395
Arg413
Arg413
O
O
O
O
strain
strain
HO
HO
H
S
H
S
R1
R1
N
H
N H
N
N
N
N
N
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N
Acyl anion
Enamine
1.
2.
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Figure 4. Proposed formation mechanism of the tetrahedral
intermediate in MenD catalysis. The strong interaction at the
terminal carboxylate is proposed to restrict the rotation
around C2-C2α so that no hydrogen bond is formed between
C2α-OH and N4’ of the cofactor throughout the reaction pro-
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ASSOCIATED CONTENT
Supporting Information
Experimental procedures and additional results in Figures S1-
S6 and Table S1-S2. This material is available free of charge
via the Internet at http://pubs.acs.org.
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